The invention relates generally to the diagnosis and treatment of neurological diseases and specifically to determining if a patient""s microglial and/or astrocyte cells are expressing apoE and suppressing such expression.
A number of important neurological diseases including Alzheimer""s disease (AD), cerebral amyloid angiopathy (CAA), and prion-mediated diseases are characterized by the deposition of aggregated proteins, referred to as amyloid, in the central nervous system (CNS) (for reviews, see Glenner et al. (1989) J. Neurol. Sci. 94:1-28; Haan et al. (1990) Clin. Neurol. Neurosurg. 92(4):305-310. These highly insoluble aggregates are composed of nonbranching, fibrillar proteins with the common characteristic of a xcex2-pleated sheet conformation. In the CNS, amyloid can be present in cerebral and meningeal blood vessels (cerebrovascular deposits) and in brain parenchyma (plaques). Neuropathological studies in human and animal models indicate that cells proximal to amyloid deposits are disturbed in their normal functions (Mandybur (1989) Acta Neuropathol. 78:329-331; Kawai et al. (1993) Brain Res. 623:142-6; Martin et al. (1994) Am. J. Pathol. 145:1348-1381; Kalaria et al. (1995) Neuroreport 6:477-80; Masliah et al. (1996) J. Neurosci. 16:5795-5811). AD studies additionally indicate that amyloid fibrils may actually initiate neurodegeneration (Lendon et al. (1997) J. Am. Med. Assoc. 277:825-31; Yankner (1996) Nat. Med. 2:850-2; Selkoe (1996) J. Biol. Chem. 271:18295-8; Hardy (1997) Trends Neurosci. 20:154-9).
AD and CAA share biochemical and neuropathological markers, but differ somewhat in the extent and location of amyloid deposits as well as in the symptoms exhibited by affected individuals. The neurodegenerative process of AD, the most common cause of progressive intellectual failure in aged humans, is characterized by the progressive and irreversible deafferentation of the limbic system, association neocortex, and basal forebrain accompanied by neuritic plaque and tangle formation (for a review see Terry et al. (1994) xe2x80x9cStructural alteration in Alzheimer""s disease.xe2x80x9d In: Alzheimer""s disease (Terry et al. eds.), pp. 179-196. Raven Press, New York). Dystrophic neurites, as well as reactive astrocytes and microglia, are associated with these amyloid-associated neurite plaques. Although, the neuritic population in any given plaque is mixed, the plaques generally are composed of spherical neurites that contain synaptic proteins, APP (type I), and fusiform neurites containing cytoskeletal proteins and paired helical filaments (PHF; type II).
CAA patients display various vascular syndromes, of which the most documented is cerebral parenchymal hemorrhage. Cerebral parenchymal hemorrhage is the result of extensive amyloid deposition within cerebral vessels (Hardy (1997) Trends Neurosci. 20:154-9; Haan et al. (1990) Clin. Neurol. Neurosurg. 92:305-10; Terry et al., supra; Vinters (1987) Stroke 18:211-24; Itoh et al. (1993) J. Neurological Sci. 116:135-41; Yamada et al. (1993) J. Neurol. Neurosurg. Psychiatry 56:543-7; Greenberg et al. (1993) Neurology 43:2073-9; Levy et al. (1990) Science 248:1124-6). In some familial CAA cases, dementia was noted before the onset of hemorrhages, suggesting the possibility that cerebrovascular amyloid deposits may also interfere with cognitive functions.
In both AD and CAA, the main amyloid component is the amyloid xcex2 protein (Axcex2). The Axcex2 peptide, which is generated from the amyloid xcex2 precursor protein (APP) by two putative secretases, is present at low levels in the normal CNS and blood. Two major variants, Axcex21-40 and Axcex21-42, are produced by alternative carboxy-terminal truncation of APP (Selkoe et al. (1988) Proc. Natl. Acad. Sci. USA 85:7341-7345; Selkoe, (1993) Trends Neurosci 16:403-409). Axcex21-42 is the more fibrillogenic and more abundant of the two peptides in amyloid deposits of both AD and CAA. In addition to the amyloid deposits in AD cases described above, most AD cases are also associated with amyloid deposition in the vascular walls (Hardy (1997), supra; Haan et al. (1990), supra; Terry et al., supra; Vinters (1987), supra; Itoh et al. (1993), supra; Yamada et al. (1993), supra; Greenberg et al. (1993), supra; Levy et al. (1990), supra). These vascular lesions are the hallmark of CAA, which can exist in the absence of AD.
The precise mechanisms by which neuritic plaques are formed and the relationship of plaque formation to the AD-associated, and CAA-associated neurodegenerative processes are not well-defined. However, evidence indicates that dysregulated expression and/or processing of APP gene products or derivatives of these gene products derivatives are involved in the pathophysiological process leading to neurodegeneration and plaque formation. For example, missense mutations in APP are tightly linked to autosomal dominant forms of AD (Hardy (1994) Clin. Geriatr. Med. 10:239-247; Mann et al. (1992) Neurodegeneration 1:201-215). The role of APP in neurodegenerative disease is further implicated by the observation that persons with Down""s syndrome who carry an additional copy of the human APP (hAPP) gene on their third chromosome 21 show an overexpression of hAPP (Goodison et al. (1993) J. Neuropathol. Exp. Neurol. 52:192-198; Oyama et al. (1994) J. Neurochem. 62:1062-1066) as well as a prominent tendency to develop AD-type pathology early in life (Wisniewski et al. (1985) Ann. Neurol. 17:278-282). Mutations in Axcex2 are linked to CAA associated with hereditary cerebral hemorrhage with amyloidosis (Dutch (HCHWA-D)(Levy et al. (1990), supra), in which amyloid deposits preferentially occur in the cerebrovascular wall with some occurrence of diffuse plaques (Maat-Schieman et al. (1994) Acta Neuropathol. 88:371-8; Wattendorff et al. (1995) J. Neurol. Neurosurg. Psychiatry 58:699-705). A number of hAPP point mutations that are tightly associated with the development of familial AD encode amino acid changes close to either side of the Axcex2 peptide (for a review, see, e.g., Lannfelt et al. (1994) Biochem. Soc Trans. 22:176-179; Clark et al. (1993) Arch. Neurol. 50:1164-1172). Finally, in vitro studies indicate that aggregated Axcex2 can induce neurodegeneration (see, e.g., Pike et al. (1995) J. Neurochem. 64:253-265).
More recently, the apoE protein has been implicated in Alzheimer""s disease (hereafter xe2x80x9cADxe2x80x9d) and cognitive performance. Saunders et al. Neurol. 43:1467-1472 (1993); Corder et al. Science 261:921-923 (1993); and Reed et al. Arch. Neurol. 51:1189-1192 (1994). Apolipoprotein E (ApoE) is a 34,000 molecular weight protein which is the product of a single gene on chromosome 19. ApoE-containing lipoproteins are found in the cerebrospinal fluid and appear to play a major role in lipid transport in the central nervous system (CNS). Pitas et al. J. Biol. Chem. 262:14352-14360 (1987). ApoE mRNA is abundant in the brain, where it is synthesized and secreted primarily by astrocytes. Elshourbagy et al. Proc. Natl. Acad. Sci USA 82:203-207 (1985); Boyles et al. J. Clin. Invest. 76:1501-1513 (1985); and Pitas et al. Biochem. Biophys. Acta 917:148-161 (1987). The liver, followed by the brain, has the highest level of apoE mRNA expression in the human body. In normal brains, the major source of apoE is from astrocytes. The source of apoE in senile plaques, however, remains unclear. El Khoury at al., Neurobiol. Aging 19:S81-S84 (1998); Boyles et al., J. Clin. Invest. 76:1501-1513 (1985).
ApoE levels dramatically increase (about 250-fold) after peripheral nerve injury. Mxc3xcller et al. Science 228:499-501 (1985); and Ignatius et al. Proc. Natl. Acad. Sci. USA 83:1125-1129 (1986). For CNS neuronal repair, regulation appears to occur in response to neuronal injury, although it is not clear whether the apoE secreted in response to injury is produced by neurons or by glia. (Messer-Joudrier et al., Eur. J. Neurosci. 8:265-261 (1996).
Human apoE exists in three major isoforms designated apoE2, apoE3 and apoE4 (for review, see Mahley (in press) Molecular and Genetic Bases of Neurological Disease 2nd ed.; and Mahley Science 240:622-630 (1988)). The different isoforms result from amino acid substitutions at amino acid residue positions 112 and 158. The common isoform, apoE3, has a cysteine residue at position 112 and an arginine residue at position 158. The apoE4 isoform differs from apoE3 only at position 112, which is an arginine residue. The apoE2 isoform, associated with type III hyperlipoproteinemia (Mahley (1988)), differs from apoE3 only at position 158, which is a cysteine residue. ApoE3 and apoE4 bind normally to the low density lipoprotein (LDL) receptor, whereas apoE2 binds weakly.
The apoE4 allele is associated with the two characteristic neuropathologic lesions of AD extracellular neuritic plaques representing deposits of amyloid beta (Axcex2) peptide and intracellular neurofibrillary tangles representing filaments of hyperphosphorylated tau, a microtubule-associated protein. For review, see, McKhann et al. Neurol. 34:939-944 (1984); Selkoe Neuron 6:487-498 (1991); Crowther Curr. Opin. Struct. Biol. 3:202-206 (1993); Roses Curr. Neurol. 14:111-141 (1994); Weisgraber et al. Curr. Opin. Lipidol. 5:110-116 (1994); and Weisgraber et al. Curr. Opin. Struct. Biol. 4:507-515 (1994).
There are currently no effective therapies for arresting, preventing and, more importantly, reversing the impairment of central nervous system function once a degenerative cascade begins. Likewise, there is no current therapy for restoration of normal, central nervous system function when the induced stress has a less catastrophic or partially reversible effect compared to the dementias. The effects of events that impair the function of the CNS, such as traumatic brain injury and stroke are in need of such therapies to mitigate or reverse the resulting damage.
A method is disclosed which comprises: (1) analyzing glial cells (astrocytes and microglial cells) to determine if the cells are expressing apoE and if expression is found; and (2) administering a compound which reduces or completely eliminates apolipoprotein E (apoE) expression by glial cells. The present invention provides a method of arresting, preventing and/or reversing the impairment of central nervous system function by reducing xcex2-amyloid plaque burden via the administration of compounds that reduce apoE production. Exemplary compounds that can be used in the method of the invention are: 1) inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase; 2) inhibitors of cholesterol biosynthesis; 3) inhibitors of protein isoprenylation, preferably inhibitors of gerangeranylation; and/or 4) inhibitors of NF-xcexaB activation or function. The compounds used in the method of the invention, alone or in combination, reduce the release of apoE and thereby reduce the rate of formation of xcex2-amyloid plaque in brain tissue.
In one embodiment of the invention, the compound administered is characterized by an ability to reduce the activity of 3-hydroxy-3-methylglutanyl coenzyme A (HMG CoA) reductase in brain tissue. Preferably, the molecules are compounds in the Statin family, and more preferably the compound is mevastatin, lovastatin, compactin, nisvastatin, atorvastatin, pravastatin, simvastatin, or fluvastatin.
In another embodiment of the invention, the compound administered is characterized by an ability to reduce protein isoprenylation. The compound may reduce protein isoprenylation such as geranylation, famesylation, and more preferably suppresses geranylgeranylation. In one preferred embodiment, the compound suppresses enzymes involved in geranylgeranylatyion, e.g., inhibitors of geranylgeranyl protein transferase (GGPTase) and/or geranylgeranyldipiphosphate (GGPP) synthase.
In yet another embodiment of the invention, the compound administered to reduce the xcex2-amyloid plaque burden in a subject suppresses cholesterol biosynthesis, which reduces the production of apoE in brain tissue of the treated subject. Preferably, the compounds used to suppress cholesterol biosynthesis are inhibitors of biosynthesis and/or the mevalonate pathways.
In yet another embodiment of the invention, the compound administered to reduce the xcex2-amyloid plaque burden in a subject inhibits the activation or function of NF-xcexaB. Such a compound may, for example, inhibit NF-xcexaB directly (e.g., by binding-to the activated molecule), inhibit release of NF-xcexaB by IxcexaB, or inhibit activity of one or more molecules involved the kinase cascade that mediates NK-xcexaB activation and translocation to the nucleus.
An object of the invention is to reduce the level of xcex2-amyloid plaques in the CNS of a mammal by reducing levels of HMG CoA reductase activity, by reducing NF-xcexaB activity, and/or by reducing protein isoprenylation, each of which alone or in combination results in reduced production of apoE in brain tissue. Preferably, the compound used to reduce HMG CoA reductase activity is an HMG CoA reductase inhibitor, and more preferably the compound is in the Statin family or a compound which inhibits protein isoprenylation, e.g., an inhibitor of geranylgeranylation.
Another object of the invention is to treat a subject with an amyloid-associated disorder by administering to the subject a compound that reduces HMG CoA reductase activity, NF-xcexaB activation or activity, or isoprenylation, e.g., geranylgeranylation. This in turn suppresses the release of apoE in the brain, reducing the level of amyloid plaque production in brain tissue. This treatment may be used in any amyloid-associated disorder, preferably CAA or a prion-mediated disorder, and more preferably AD.
A feature of the invention is that it is effective in treating individuals with an ApoE4 allele, but is also effective in treating ApoE3 and ApoE2 individuals.
Another object of the invention is to reduce the formation of amyloid plaques in the brain of a subject at risk for an amyloid associated disorder by administering to the subject a compound that reduces HMG CoA reductase activity. Reduction in HMG CoA reductase activity results in reduced xcex2-amyloid stimulated release of apoE from cells, and preferably reduces release of apoE from glial cells.
Another object of the invention is to prevent the formation of amyloid plaques in the brains of subjects by administering a compound which inhibits isoprenylation of proteins.
Another object of the invention is to prevent the formation of amyloid plaques in the brains of subjects by administering a compound which inhibits NF-xcexaB activation and/or function.
A feature of the invention is that it is particularly effective for people at risk for amyloid disorders due to the presence of the apoE4 allele.
An advantage of the invention is that many of the identified compounds, e.g.,HMG CoA reductase inhibitors, are safe for treatment for extended periods of time.
Another advantage of the invention is that compounds of the invention are able to cross the blood brain barrier at concentrations sufficient to elicit a pharmacological effect.
These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the invention as more fully described below.